Method for the safe induction of immunity against rsv

ABSTRACT

Methods of inducing a safe immune response against respiratory syncytial virus (RSV) in a human subject in need thereof, including administering to the subject a composition including recombinant adenovirus including a nucleic acid encoding an RSV Fusion (F) protein including the amino acid sequence of SEQ ID NO: 1, and a pharmaceutically acceptable carrier, in a total dose of from about 1×10 10  to about 2×10 11  viral particles (vp), are described.

FIELD OF THE INVENTION

The invention relates to methods for safely inducing effective immunityagainst RSV. In particular, the invention relates to adenovirus serotype26 expression vectors expressing RSV F protein providing the safeinduction of immunity against multiple strains of RSV in human subjects.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a highly contagious childhoodpathogen of the respiratory tract which is believed to be responsiblefor ˜200,000 childhood deaths annually. In children younger than 2years, RSV accounts for approximately 50% of the hospitalizations due torespiratory infections, with a peak of hospitalization occurring at 2-4months of age. It has been reported that almost all children will haveexperienced infection with RSV by the age of two, and repeated infectionduring life is attributed to low natural immunity. In the elderly, theRSV disease burden is similar to those caused by non-pandemic influenzaA infections.

RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Itsgenome encodes for various proteins, including the membrane proteinsknown as RSV Glycoprotein (G) and RSV fusion (F) protein which are themajor antigenic targets for neutralizing antibodies.

Unlike the RSV G protein, the F protein is conserved between RSVstrains; which makes it an attractive vaccine candidate able to elicitbroadly neutralizing antibodies. The F protein is a transmembraneprotein and it is incorporated in the virion membrane from cellularmembrane during virus budding. The RSV F protein facilitates infectionby fusing the viral and host-cell membranes. In the process of fusion,the F protein refolds irreversibly from a labile pre-fusion conformationto a stable post-fusion conformation. The protein precursor, F0,requires cleavage during intracellular maturation by a furin-likeprotease. There are two furin sites, cleavage of which results inremoval of a p27 peptide and formation of two domains: an N-terminal F2domain and a C-terminal F1 domain (FIG. 1). The F 2 and F1 domains areconnected by two cystine bridges. Antibodies against the fusion proteincan prevent virus uptake in the cell and thus have a neutralizingeffect. Besides being a target for neutralizing antibodies, RSV Fcontains cytotoxic T cell epitopes (Pemberton et al, 1987, J. Gen.Virol. 68: 2177-2182).

Despite 50 years of research, there is still no licensed vaccine againstRSV. One major obstacle to the vaccine development is the legacy ofvaccine-enhanced disease in a clinical trial in the 1960s with aformalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children werenot protected against natural infection and infected childrenexperienced more severe illness than non-vaccinated children, includingtwo deaths. This phenomenon is referred to as ‘enhanced disease’.

Since the trial with the FI-RSV vaccine, various approaches to generatean RSV vaccine have been pursued. Attempts include classical liveattenuated cold passaged or temperature sensitive mutant strains of RSV,(chimeric) protein subunit vaccines, peptide vaccines and RSV proteinsexpressed from recombinant viral vectors, including adenoviral vectors.Although some of these vaccines showed promising pre-clinical data, novaccine has been licensed for human use due to safety concerns or lackof efficacy.

The most potent RSV neutralizing antibodies bind to a particular site(site zero) on the RSV F protein which is only exposed when the RSVprotein is in its pre-fusion conformation which makes this particularconformation very attractive as a vaccine antigen. However, the Fprotein in its pre-fusion conformation is very unstable and readilyundergoes a major conformational shift to the post-fusion conformation.Due to its instability the pre-fusion conformation thus has a propensityto prematurely refold into the stable postfusion conformation. Thisphenomenon is an intrinsic feature of the protein both in solution andon the surface of the virions.

Vaccine candidates based on the RSV F protein have failed due toproblems with e.g. stability, purity, reproducibility, and potency.Indeed, despite many efforts to produce a vaccine against RSV thatcontain pre-fusion forms of RSV F protein, no stable pre-fusion RSV Fpolypeptides have been described that have been tested in humans.

Therefore, a need still exists for safe and effective vaccines andmethods of vaccinating against RSV. The present invention aims atproviding such methods for vaccinating against RSV in a safe manner.

SUMMARY OF THE INVENTION

According to the present invention it has for the first time beendemonstrated that recombinant adenovectors expressing RSV F proteinstabilized in the pre-fusion comformation induce a safe and effective(immunogenic) immune response against RSV when administered to humansubjects.

The invention thus provides a method of inducing a safe immune responseagainst respiratory syncytial virus (RSV) in a human subject in needthereof, comprising administering to the subject a compositioncomprising recombinant adenovirus comprising nucleic acid encoding anRSV Fusion (F) protein comprising the amino acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable carrier, in a total dose of fromabout 1×10¹⁰ to about 2×10¹¹ viral particles (vp) of adenovirus.

In a further aspect, the invention provides a vaccine compositioncomprising recombinant adenovirus comprising nucleic acid encoding anRSV Fusion (F) protein comprising the amino acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable carrier, in a total dose of about1×10¹⁰ to about 2×10¹¹ viral particles (vp), for use in a method ofinducing a safe immune response against respiratory syncytial virus(RSV) in a subject in need thereof.

In certain embodiments of the invention, the total dose is from about5×10¹⁰ to about 1×10¹¹ viral particles (vp) of adenovirus. In certainembodiments, the dose is about 5×10¹⁰ or about 1×10¹¹ vp of adenovirus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the RSV F protein. A proteinprecursor includes F2 and F1 domains and p27 peptide that is removedfrom the mature proteins by cleavage with furin-like proteases. Thecleavage sites are indicated by arrows. The numbers on top of the boxesindicate amino acid positions in the full length protein excludingsignal peptide. In the F1, structural elements are shown: fusion peptide(FP), refolding region 1 (RR1) including heptad repeat A (HRA) andrefolding region 2 (RR2) including heptad repeat B (HRB).

FIG. 2: Relative surface expression of F protein variants. Full lengthvariants of F protein were expressed in HEK293T cells. The cell werestained with anti-RSV F antibody (CR9503) and analyzed by Flow Cytometry(FACS). The Mean Fluorescent Intensity (MFI) values were calculated andnormalized to MFI of control F wild type (Fwt)-transfected cell sample.The MFI of Fwt was set to 1. Bars represent mean values, error barsrepresent range of values.

FIG. 3: Fraction of pre-fusion F protein on cell surface. Full lengthvariants of F protein were expressed in HEK293T cells. The cell werestained with anti-RSV F antibody CR9503 and anti-pre-fusion RSV Fantibody CR9501 and analyzed by Flow Cytometry. The Mean FluorescentIntensity (MFI) values were calculated and MFI measured by CR9501 wasnormalized to MFI measured by CR9503. The normalized MFI values indicatefraction of the pre-F on the cell surface. Bars represent mean values,error bars represent range of values.

FIG.4: Relative surface expression of F protein variants. Solubleversions with the transmembrane region and cytoplasmic region deleted(Fs1) and full length variants of F protein were expressed in HEK293Tcells. The expression level of the soluble protein was measured in theculture supernatant by octet and the full length variants were tested bycell staining using anti-RSV F antibody (CR9503) and analyzed by FlowCytometry. The Mean Fluorescent Intensity (MFI) values were calculatedand normalized to MFI of control F wild type (Fwt)-transfected cellsample. The MFI of Fwt was set to 1. Bars represent mean values, errorbars represent range of values.

FIG. 5: Temperature stability of the F protein variants. Full lengthvariants of F protein were expressed in HEK293T cells. After heat-shock,the cell were stained with anti-RSV F antibody (CR9501—solid lines andCR9503—dashed lines) and analyzed by Flow Cytometry. The percentage ofcells positive for the staining was determined. Symbols represent meanvalues, error bars represent range of values. (A) The percentage ofcells positive for the staining was determined. (B) The Mean FluorescentIntensity (MFI) values were calculated and normalized to MFI of 37C cellsample. The MFI of 37° C. sample was set to 1. Dotted lines correspondto background staining at 60° C.

FIG. 6: Stability of the F proteins. PreF is more stable than FA2protein in prefusion conformation on the cell surface in the heat-stressassay. A549 cells were infected with 26 and Ad35 comprising the insertFA2 (wt RSV F, grey bars) or prefusion F stabilized insert (preF2.2,black bars) at the indicated MOI. The cells were temperature treated atthe indicated temperature for 15 minutes before staining. Top:percentage of cells presenting prefusion F on their surface (detected byCR9501 antibody); bottom: percentage of cells presenting any form of theF protein, prefusion and post fusion (detected by CR9503 antibody). Thevalues were normalized to 37° C. samples. All bars represent a singlemeasurement.

FIG. 7: Ad26.RSV.preF2.1 and F2.2 elicit a cellular immune responseafter single administration in mice. Horizontal bars depict thegeometric mean of the response within a group. The background level iscalculated as the 95% percentile of the spot forming units (SFU)observed in non-stimulated splenocytes, and is indicated with a dottedline.

FIG. 8: Ad26.RSV.preF2.1 and F2.2 induce increased virus neutralizingantibodies when compared to Ad26.RSV.FA2, after single immunization inmice. Balb/c mice (n=4 per group) were immunized with the indicated doseof 108 to 1010 viral particles (vp) Ad26.RSV.FA2 or Ad26.RSV.preF2.1 orAd26.RSV.preF2.2, or with formulation buffer, and humoral immuneresponses were assayed in the serum isolated 8 weeks after immunization.(A) Virus neutralizing antibodies were determined against RSV A Longusing a micro-neutralization assay with an ELISA based read out. Titersare given as the log2 value of the IC50. (B)Pre-fusion or post-fusion Fantibody titers were determined by ELISA, and the ratio between pre- andpost-fusion F antibodies for all samples that showed pre- andpost-fusion F titers above lower limit of quantification (LLoQ) wascalculated. (C) Subclass ELISA was performed using post-fusion RSV F A2as coating reagent, and the IgG2a/IgG1 ratio (log10) is plotted. Ratio'sobserved for Th1 (serum derived from animals immunized with RSV Fexpression Adenoviral vectors) and Th2 (serum derived from FI-RSVimmunized animals) references samples are indicated with dashed lines.The LLoQ is indicated with a dotted lines (panels A), and horizontalbars represent the mean responses per group.

FIG. 9: Ad26.RSV.preF2.2 elicits antibody responses that neutralize awide range of RSV isolates. Sera from Balb/c mice that were immunizedwith 1010 viral particles (vp) Ad26.RSV.FA2 (n=3) or Ad26.RSV.preF (n=4)or formulation buffer (n=2) were used in virus neutralization assays(micro-neutralization assay with an ELISA based read out) with the RSV A(upper panels) and B strains (lower panels) indicated. Titers are givenas the log2 value of the IC50, and horizontal bars represent the meanresponse per group. LLoQ is indicated with a dashed line.

FIG. 10: Single immunization with Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2at low doses protects cotton rats against challenge with the homologousRSV A2. Cotton rats (Sigmodon hispidus) (n=7 to 9 per group) wereimmunized with the indicated doses (in vp/animal) of Ad26.RSV.preF2.2,Ad26.RSV.FA2 (left panels), Ad35.RSV.preF2.2 or Ad35.RSV.FA2 (rightpanels) by single intramuscular administration. Control immunizationswere performed with formulation buffer, FI-RSV, or intranasalapplication of a low dose of RSV A2. At seven weeks post-immunizationanimals were challenged intranasally with 105 pfu RSV A2. The lung (A-B)and nose viral titers (C-D) were determined by plaque assay 5 days afterchallenge. (E-F) Sera taken just before challenge were used to perform avirus neutralizing assay with the RSV A Long strain(micro-neutralization assay with an ELISA based read out). The dottedline represents the lower level of quantification (LLoQ). Horizontalbars represent the mean titer per group.

FIG. 11: Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 immunization of cottonrats does not result in increased alveolitis scores after RSV A2challenge. Cotton rats (Sigmodon hispidus) (n=7 to 9 per group) wereimmunized with the indicated doses (in vp/animal) of Ad26.RSV.preF2.2,Ad26.RSV.FA2 (upper panel), Ad35.RSV.preF2.2, or Ad35.RSV.FA2 (lowerpanel) by single intramuscular administration. Control immunizationswere performed with formulation buffer, FI-RSV, or intranasalapplication of a low dose of RSV A2. At seven weeks post-immunizationanimals were challenged intranasally with 105 pfu RSV A2. Alveolitis wasscored by histopathological examination of one lung lobe 5 days afterchallenge on a non-linear scale from 0 to 4. The horizontal dotted linemarks the maximal score of the control animals that were pre-exposed toRSV-A2 before challenge to mimic a natural exposure to RSV that does notlead to ERD.

DETAILED DESCRIPTION OF THE INVENTION

Although respiratory syncytial virus (RSV) infects people throughoutlife, most people fail to mount a long lasting protective immuneresponse. In addition, in the elderly the waning immune responsecontributes to increased susceptibility to severe disease after RSVinfection causing significant morbidity and mortality. There areindications in the literature that both neutralizing antibody and T-cellmediated protection play a role in preventing RSV infection. It istherefore believed that a successful RSV vaccine, in particular for theelderly, should increase both potent neutralizing antibody levels andinduce a robust T-cell response.

RSV infection induces virus specific antibodies which are mainlydirected against the fusion (F) protein. The most potent RSVneutralizing antibodies found in human sera bind to a particular site(site ϕ or site zero) on the RSV F protein, which is only exposed whenthe RSV protein is in its pre-fusion conformation which makes thisparticular conformation very attractive as a vaccine antigen. However,the F protein in its pre-fusion conformation is very unstable andreadily undergoes a major conformational shift to the post-fusionconformation.

In the research that has led to the present invention, a stabilized RSVF protein was prepared with a unique set of amino acid mutationscompared to the wild type RSV F antigen from the RSV A2 strain (GenbankACO83301.1). By demonstrating specific binding in vitro to pre-fusionspecific antibodies it was shown that indeed a stabilized RSV F antigenin the pre-fusion conformation was obtained.

According to the present invention, it has now been shown that thestabilized RSV F protein maintains its pre-fusion conformation in vivo,is immunogenic and can be safely administered to human subjects in needthereof.

Thus, according to the present invention methods are provided forinducing a safe immune response against RSV in a human subject in needthereof, comprising administering to the subject a compositioncomprising recombinant adenovirus comprising nucleic acid encoding anRSV Fusion (F) protein comprising the amino acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable carrier, in a total dose of fromabout 1×10¹⁰ to about 2×10¹¹ viral particles (vp) of adenovirus.

In a further aspect, the invention provides a vaccine compositioncomprising recombinant adenovirus comprising nucleic acid encoding anRSV Fusion (F) protein comprising the amino acid sequence of SEQ ID NO:1, and a pharmaceutically acceptable carrier, in a total dose of about1×10¹⁰ to about 2×10¹¹ viral particles (vp), for use in a method ofinducing a safe immune response against respiratory syncytial virus(RSV) in a subject in need thereof.

In certain embodiments of the invention, the composition comprisesrecombinant adenovirus comprising nucleic acid encoding an RSV Fusion(F) protein comprising the amino acid sequence of SEQ ID NO: 1, and apharmaceutically acceptable carrier, in a total dose of from about5×10¹⁰ to about 1×11 viral particles (vp) of adenovirus.

In certain embodiments, the dose is about 5×10¹⁰ or about 1×10¹¹ vp ofadenovirus.

According to the present invention it has been shown that adenovirusencoding RSV F protein in the pre-fusion conformation (in particular theRSV F protein of SEQ ID NO: 1) is highly immunogenic. Humoral immuneresponses are increased as compared to similar doses of adenovirusencoding the wild-type RSV F protein, which is not stabilized in thepre-fusion conformation and likely quickly transitions into thepost-fusion conformation upon expression.

In certain embodiments, the immune response comprises the induction ofantibodies directed against RSV F protein. Thus, according to certainembodiments, the immune response comprises the induction of antibodiesthat specifically bind to the RSV F protein, as measured in an ELISA.

In certain embodiments, the immune response comprises the induction ofRSV neutralizing antibodies. In certain embodiments, the neutralizingantibodies are capable of neutralizing RSV A and B strains. In certainembodiments, the immune response comprises the induction of antibodiesthat are capable of neutralizing RSV A and B strains in a VNA assay.

As used herein the “ induction of antibodies” means that the level ofantibodies as measured after administration of composition comprisingrecombinant adenovirus expressing the RSV F protein of SEQ ID NO: 1 ishigher than the level of antibodies prior to administration of saidcomposition.

In certain embodiments, the immune response comprises the induction ofantibodies specific for the RSV F protein in the pre-fusion conformationand antibodies specific for the RSV F protein in the post-fusionconformation, wherein the geometric mean titer (GMT) increase ofantibodies specific for RSV F protein in the pre-fusion conformation ishigher than the GMT increase of antibodies specific for RSV F protein inthe post-fusion conformation, in enzyme linked immunosorbent assays(ELISAs). The methods of the present invention thus result in a largerincrease of antibodies that are specifically binding to RSV F protein inthe pre-fusion conformation as compared to antibodies that arespecifically binding to RSV F protein in the post-fusion conformation.Without wishing to be bound to a particular theory, it is believed thatthis results in a more effective immune response since pre-fusionspecific antibodies are thought to be more effective in neutralizing RSVvirus, and thereby more effective in prevention RSV infection (Gilman MS, Castellanos C A, Chen M, Ngwuta J O, Goodwin E, Moin S M, Mas V,Melero J A, Wright P F, Graham B S, McLellan J S, Walker L M. SciImmunol. 2016 Dec. 16;1(6). pii: eaaj1879. doi:10.1126/sciimmunol.aaj1879. Epub 2016 Dec. 9.Rapid profiling of RSVantibody repertoires from the memory B cells of naturally infected adultdonors) In certain embodiments, the ratio between the GMT increase ofpost-fusion F specific antibodies as measured in ELISA and the meangeometric titer increase of neutralizing antibodies as measured in a VNAassay is reduced after administration of said composition as compared tosaid ratio before administration of said composition. The methods of thepresent invention thus results in a more favourable composition of theRSV specific humoral response that favours neutralizing antibodies overbinding antibodies.

In certain embodiments, the immune response further comprises a cellularresponse as indicated by IFNgamma producing T cells as measured in anIFNy ELISPOT in response to stimulation with a pool of peptides coveringthe F protein of SEQ ID NO: 1 and/or by measurement of CD4 and CD8T-cell subsets expressing IFNγ, IL-2 and TNFα by intracellular staining(ICS) after stimulation with a pool of peptides covering the RSV Fprotein of SEQ ID NO: 1. It has been suggested that RSV specific T cellscan support the prevention of infection and limit disease; this could beespecially benificial for the older adults since it has been describedthat the cellular response may decrease with age (Openshaw P J M, ChiuC, Culley F J, Johansson C. Annu Rev Immunol. 2017 Apr. 26;35:501-532.doi: 10.1146/annurev-immunol-051116-052206. Epub 2017 Feb. 6. Protectiveand Harmful Immunity to RSV Infection.)

By inducing a safe and immunogenic immune response, the methods of thepresent invention can be used to prevent serious lower respiratory tractdisease leading to hospitalization and to decrease the frequency ofcomplications such as pneumonia and bronchiolitis due to RSV infectionand replication in a subject.

As used herein, the terms nucleic acid, nucleic acid molecule, nucleicacid or nucleotide sequence, and polynucleotide are used interchangeablyand all refer to the linear biopolymers (chains) made from nucleotides,including DNA and RNA. It will be understood by a skilled person thatnumerous different nucleic acid molecules can encode the samepolypeptide as a result of the degeneracy of the genetic code. It isalso understood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described there to reflect the codonusage of any particular host organism in which the polypeptides are tobe expressed. Therefore, unless otherwise specified, a “nucleic acidmolecule encoding an amino acid sequence” includes all nucleotidesequences that are degenerate versions of each other and that encode thesame amino acid sequence. Nucleotide sequences that encode proteins andRNA may include introns. Sequences herein are provided from 5′ to 3′direction, as custom in the art.

In certain embodiments, the nucleic acid molecule encoding the RSVpre-fusion F protein are codon optimized for expression in mammaliancells, such as human cells. Methods of codon-optimization are known andhave been described previously (e.g. WO 96/09378). In a preferredembodiment, the nucleic acid molecule encoding the RSV pre-fusion Fprotein comprises the nucleic acid sequence of SEQ ID NO: 2. In certainembodiments, the nucleic acid encoding the RSV F protein consists of thenucleic acid sequence of SEQ ID NO: 2.

According to the invention, the subject in need thereof is a humansubject. Preferably, the subject is an elderly subject, i.e. a human of60 years and older. According to the present invention it has been shownthat administration of recombinant adenovirus comprising nucleic acidencoding the RSV F protein in the pre-fusion conformation is safe andimmunogenic, i.e. results in a potent humoral and cellular response,also in older subjects which may have a weakened immune system.

In certain embodiments, the recombinant adenovirus (also referred to asadenoviral vector) is a human recombinant adenovirus. The preparation ofrecombinant adenoviral vectors is well known in the art. The term‘recombinant’ for an adenovirus, as used herein implicates that it hasbeen modified by the hand of man, e.g. it has altered terminal endsactively cloned therein and/or it comprises a heterologous gene, i.e. itis not a naturally occurring wild type adenovirus.

In certain embodiments, the recombinant adenovirus according to theinvention is deficient in at least one essential gene function of the E1region, e.g. the E1 a region and/or the E1 b region, of the adenoviralgenome that is required for viral replication. In certain embodiments,the adenovirus according to the invention is deficient in at least partof the non-essential E3 region. In certain embodiments, the adenovirusis deficient in at least one essential gene function of the E1 regionand at least part of the non-essential E3 region. The adenoviral vectorcan be “multiple deficient,” meaning that the adenoviral vector isdeficient in one or more essential gene functions in each of two or moreregions of the adenoviral genome. For example, the aforementionedE1-deficient or E1-, E3-deficient adenoviral vectors can be furtherdeficient in at least one essential gene of the E4 region and/or atleast one essential gene of the E2 region (e.g., the E2A region and/orE2B region).

In certain embodiments, the adenovirus is a human adenovirus of theserotype 26 or 35. The vaccines according to the invention based onthese serotypes appear more potent than the ones described in the priorart that were based on Ad5, since those failed to provide completeprotection against RSV challenge replication after a singleintramuscular administration (Kim et al, 2010, Vaccine 28: 3801-3808;Kohlmann et al, 2009, J Virol 83: 12601-12610; Krause et al, 2011,Virology Journal 8:375). The serotype of the invention further generallyhas a low seroprevalence and/or low pre-existing neutralizing antibodytiters in the human population. Recombinant adenoviral vectors of theseserotypes with different transgenes are evaluated in clinical trials,and thus far shows to have an excellent safety profile. Preparation ofrAd26 vectors is described, for example, in WO 2007/104792 and in Abbinket al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO2007/104792. Preparation of rAd35 vectors is described, for example, inU.S. Pat. No. 7,270,811, in WO 00/70071, and in Vogels et al., (2003) JVirol 77(15): 8263-71. Exemplary genome sequences of Ad35 are found inGenBank Accession AC_000019 and in FIG. 6 of WO 00/70071.

A recombinant adenovirus according to the invention may bereplication-competent or replication-deficient. In certain embodiments,the adenovirus is replication deficient, e.g. because it contains adeletion in the E1 region of the genome. As known to the skilled person,in case of deletions of essential regions from the adenovirus genome,the functions encoded by these regions have to be provided in trans,preferably by the producer cell, i.e. when parts or whole of E1, E2and/or E4 regions are deleted from the adenovirus, these have to bepresent in the producer cell, for instance integrated in the genomethereof, or in the form of so-called helper adenovirus or helperplasmids. The adenovirus may also have a deletion in the E3 region,which is dispensable for replication, and hence such a deletion does nothave to be complemented.

For non-subgroup C E1-deficient adenoviruses such as Ad35 (subgroup B)or Ad26 (subgroup D), it is preferred to exchange the E4-orf6 codingsequence of these non-subgroup C adenoviruses with the E4-orf6 of anadenovirus of subgroup C such as Ad5. This allows propagation of suchadenoviruses in well known complementing cell lines that express the E1genes of Ad5, such as for example 293 cells or PER.C6 cells (see, e.g.Havenga et al, 2006, J. Gen. Virol. 87: 2135-2143; WO 03/104467,incorporated in its entirety by reference herein). In certainembodiments, an adenovirus that can be used is a human adenovirus ofserotype 35, with a deletion in the E1 region into which the nucleicacid encoding RSV F protein antigen has been cloned, and with an E4 orf6region of Ad5. In certain embodiments, the adenovirus in the vaccinecomposition of the invention is a human adenovirus of serotype 26, witha deletion in the E1 region into which the nucleic acid encoding RSV Fprotein antigen has been cloned, and with an E4 orf6 region of Ad5.

In alternative embodiments, there is no need to place a heterologousE4orf6 region (e.g. of Ad5) in the adenoviral vector, but instead theE1-deficient non-subgroup C vector is propagated in a cell line thatexpresses both E1 and a compatible E4orf6, e.g. the 293-ORF6 cell linethat expresses both E1 and E4orf6 from Ad5 (see e.g. Brough et al, 1996,J Virol 70: 6497-501 describing the generation of the 293-ORF6 cells;Abrahamsen et al, 1997, J Virol 71: 8946-51 and Nan et al, 2003, GeneTherapy 10: 326-36 each describing generation of E1deleted non-subgroupC adenoviral vectors using such a cell line).

Alternatively, a complementing cell that expresses E1 from the serotypethat is to be propagated can be used (see e.g. WO 00/70071, WO02/40665).

For subgroup B adenoviruses, such as Ad35, having a deletion in the E1region, it is preferred to retain the 3′ end of the E1B 55K open readingframe in the adenovirus, for instance the 166 bp directly upstream ofthe pIX open reading frame or a fragment comprising this such as a 243bp fragment directly upstream of the pIX start codon (marked at the 5′end by a Bsu36I restriction site in the Ad35 genome), since thisincreases the stability of the adenovirus because the promoter of thepIX gene is partly residing in this area (see, e.g. Havenga et al, 2006,J. Gen. Virol. 87: 2135-2143; WO 2004/001032, incorporated by referenceherein).

The nucleic acid encoding the RSV F protein of SEQ ID NO: 1 can beintroduced into the adenovirus for instance by standard molecularbiology techniques. It can for instance be cloned into a deleted E1 orE3 region of an adenoviral vector. The nucleic acid (or transgene) isgenerally operably linked to expression control sequences. This can forinstance be done by placing the nucleic acid encoding the RSV F proteinunder the control of a promoter. Further regulatory sequences may beadded. Many promoters can be used for expression of a transgene(s), andare known to the skilled person. A non-limiting example of a suitablepromoter for obtaining expression in eukaryotic cells is a CMV-promoter(U.S. Pat. No. 5,385,839), e.g. the CMV immediate early promoter, forinstance comprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter. A polyadenylation signal, for example the bovinegrowth hormone polyA signal (U.S. Pat. No. 5,122,458), may be presentbehind the transgene(s).

In certain embodiments, the recombinant adenovectors of the inventioncomprise as the 5′ terminal nucleotides the nucleotide sequence:CTATCTAT. These embodiments are advantageous because such vectorsdisplay improved replication in production processes, resulting inbatches of adenovirus with improved homogeneity, as compared to vectorshaving the original 5′ terminal sequences (generally CATCATCA) (see alsoPCT publication no. WO 2013/135615 and U.S. Pat. No. 8,932,607),incorporated in its entirety by reference herein.

In certain embodiments the compositions comprising the recombinantadenovirus may further comprise, or are administered together with, oneor more adjuvants. Adjuvants are known in the art to further increasethe immune response to an applied antigenic determinant, andpharmaceutical compositions comprising adenovirus and suitable adjuvantsare for instance disclosed in WO 2007/110409, incorporated by referenceherein. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the RSV prefusion F proteins ofthe invention. Examples of suitable adjuvants include aluminium saltssuch as aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, and the like. It is also possible to use vector-encodedadjuvant, e.g. by using heterologous nucleic acid that encodes a fusionof the oligomerization domain of C4-binding protein (C4bp) to theantigen of interest (e.g. Solabomi et al, 2008, Infect Immun 76:3817-23). In certain embodiments the compositions of the inventioncomprise aluminium as an adjuvant, e.g. in the form of aluminiumhydroxide, aluminium phosphate, aluminium potassium phosphate, orcombinations thereof, in concentrations of 0.05-5 mg, e.g. from0.075-1.0 mg, of aluminium content per dose.

Administration of the compositions can be performed using standardroutes of administration. Non-limiting embodiments include parenteraladministration, such as by injection e.g. intradermal, intramuscular,etc, or subcutaneous, transcutaneous, or mucosal administration, e.g.intranasal, oral, and the like. Intranasal administration has generallybeen seen as a preferred route for vaccines against RSV. The mostimportant advantage of the live intrasal strategy is the directstimulation of local respiratory tract immunity and the lack ofassociated disease enhancement. Intranasal administration is a suitablepreferred route according to the present invention as well. Theadvantage of intramuscular administration is that it is simple andwell-established. In one embodiment of the invention the composition isadministered by intramuscular injection, e.g. into the deltoid muscle ofthe arm, or vastus lateralis muscle of the thigh. The skilled personknows the various possibilities to administer a composition, e.g. avaccine in order to induce an immune response to the antigen(s) in thevaccine.

Recombinant adenovirus can be prepared and propagated in host cells,according to well known methods, which entail cell culture of the hostcells that are infected with the adenovirus. The cell culture can be anytype of cell culture, including adherent cell culture, e.g. cellsattached to the surface of a culture vessel or to microcarriers, as wellas suspension culture.

Most large-scale suspension cultures are operated as batch or fed-batchprocesses because they are the most straightforward to operate and scaleup.

Nowadays, continuous processes based on perfusion principles arebecoming more common and are also suitable (see e.g. WO 2010/060719, andWO 2011/098592, both incorporated by reference herein, which describesuitable methods for obtaining and purifying large amounts ofrecombinant adenoviruses).

Producer cells are cultured to increase cell and virus numbers and/orvirus titers. Culturing a cell is done to enable it to metabolize,and/or grow and/or divide and/or produce virus of interest according tothe invention. This can be accomplished by methods as such well known topersons skilled in the art, and includes but is not limited to providingnutrients for the cell, for instance in the appropriate culture media.Suitable culture media are well known to the skilled person and cangenerally be obtained from commercial sources in large quantities, orcustom-made according to standard protocols. Culturing can be done forinstance in dishes, roller bottles or in bioreactors, using batch,fed-batch, continuous systems and the like. Suitable conditions forculturing cells are known (see e.g. Tissue Culture, Academic Press,Kruse and Paterson, editors (1973), and R. I. Freshney, Culture ofanimal cells: A manual of basic technique, fourth edition (Wiley-LissInc., 2000, ISBN 0-471-34889-9).

A producer cell (sometimes also referred to in the art and herein as‘packaging cell’ or ‘complementing cell’ or ‘host cell’) that can beused can be any producer cell wherein a desired adenovirus can bepropagated. For example, the propagation of recombinant adenovirusvectors is done in producer cells that complement deficiencies in theadenovirus. Such producer cells preferably have in their genome at leastan adenovirus E1 sequence, and thereby are capable of complementingrecombinant adenoviruses with a deletion in the E1 region. AnyE1-complementing producer cell can be used, such as human retina cellsimmortalized by E1, e.g. 911 or PER.C6 cells (see U.S. Pat. No.5,994,128), E1-transformed amniocytes (See EP U.S. Pat. No. 1,230,354),E1-transformed A549 cells (see e.g. WO 98/39411, U.S. Pat. No.5,891,690), GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11:213-219), 293, and the like. In certain embodiments, the producer cellsare for instance HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SFcells, and the like.

Typically, the adenovirus will be exposed to the appropriate producercell in a culture, permitting uptake of the virus. Usually, the optimalagitation is between about 50 and 300 rpm, typically about 100-200, e.g.about 150, typical DO is 20-60%, e.g.40%, the optimal pH is between 6.7and 7.7, the optimal temperature between 30 and 39° C., e.g. 34-37° C.,and the optimal MOI between 5 and 1000, e.g. about 50-300. Typically,adenovirus infects producer cells spontaneously, and bringing theproducer cells into contact with rAd particles is sufficient forinfection of the cells. Generally, an adenovirus seed stock is added tothe culture to initiate infection, and subsequently the adenoviruspropagates in the producer cells. This is all routine for the personskilled in the art.

After infection of an adenovirus, the virus replicates inside the celland is thereby amplified, a process referred to herein as propagation ofadenovirus. Adenovirus infection results finally in the lysis of thecells being infected. The lytic characteristics of adenovirus thereforepermits two different modes of virus production.

The first mode is harvesting virus prior to cell lysis, employingexternal factors to lyse the cells. The second mode is harvesting virussupernatant after (almost) complete cell lysis by the produced virus(see e.g. U.S. Pat. No. 6,485,958, describing the harvesting ofadenovirus without lysis of the host cells by an external factor). It ispreferred to employ external factors to actively lyse the cells forharvesting the adenovirus.

Methods that can be used for active cell lysis are known to the personskilled in the art, and have for instance been discussed in WO 98/22588,p. 28-35. Useful methods in this respect are for example, freeze-thaw,solid shear, hypertonic and/or hypotonic lysis, liquid shear,sonication, high pressure extrusion, detergent lysis, combinations ofthe above, and the like. In one embodiment of the invention, the cellsare lysed using at least one detergent. Use of a detergent for lysis hasthe advantage that it is an easy method, and that it is easily scalable.

Detergents that can be used, and the way they are employed, aregenerally known to the person skilled in the art. Several examples arefor instance discussed in WO 98/22588, p. 29-33. Detergents can includeanionic, cationic, zwitterionic, and nonionic detergents. Theconcentration of the detergent may be varied, for instance within therange of about 0.1%-5% (w/w). In one embodiment, the detergent used isTriton X-100.

Nuclease may be employed to remove contaminating, i.e. mostly from theproducer cell, nucleic acids. Exemplary nucleases suitable for use inthe present invention include Benzonase®, Pulmozyme®, or any other DNaseand/or RNase commonly used within the art. In preferred embodiments, thenuclease is Benzonase®, which rapidly hydrolyzes nucleic acids byhydrolyzing internal phosphodiester bonds between specific nucleotides,thereby reducing the viscosity of the cell lysate. Benzonase® can becommercially obtained from Merck KGaA (code W214950). The concentrationin which the nuclease is employed is preferably within the range of1-100 units/ml. Alternatively, or in addition to nuclease treatment, itis also possible to selectively precipitate host cell DNA away fromadenovirus preparations during adenovirus purification, using selectiveprecipitating agents such as domiphen bromide (see e.g. U.S. Pat. No.7,326,555; Goerke et al., 2005, Biotechnology and bioengineering, Vol.91: 12-21; WO 2011/045378; WO 2011/045381).

Methods for harvesting adenovirus from cultures of producer cells havebeen extensively described in WO 2005/080556.

In certain embodiments, the harvested adenovirus is further purified.Purification of the adenovirus can be performed in several stepscomprising clarification, ultrafiltration, diafiltration or separationwith chromatography as described in for instance WO 05/080556,incorporated by reference herein.

Clarification may be done by a filtration step, removing cell debris andother impurities from the cell lysate. Ultrafiltration is used toconcentrate the virus solution. Diafiltration, or buffer exchange, usingultrafilters is a way for removal and exchange of salts, sugars and thelike. The person skilled in the art knows how to find the optimalconditions for each purification step. Also WO 98/22588, incorporated inits entirety by reference herein, describes methods for the productionand purification of adenoviral vectors. The methods comprise growinghost cells, infecting the host cells with adenovirus, harvesting andlysing the host cells, concentrating the crude lysate, exchanging thebuffer of the crude lysate, treating the lysate with nuclease, andfurther purifying the virus using chromatography.

Preferably, purification employs at least one chromatography step, asfor instance discussed in WO 98/22588, p. 61-70. Many processes havebeen described for the further purification of adenoviruses, whereinchromatography steps are included in the process. The person skilled inthe art will be aware of these processes, and can vary the exact way ofemploying chromatographic steps to optimize the process. It is forinstance possible to purify adenoviruses by anion exchangechromatography steps, see for instance WO 2005/080556 and Konz et al,2005, Hum Gene Ther 16: 1346-1353. Many other adenovirus purificationmethods have been described and are within the reach of the skilledperson. Further methods for producing and purifying adenoviruses aredisclosed in for example (WO 00/32754; WO 04/020971; U.S. Pat. No.5,837,520; U.S. Pat. No. 6,261,823; WO 2006/108707; Konz et al, 2008,Methods Mol Riot 434: 13-23; Altaras et al, 2005, Adv Biochem EngBiotechnol 99: 193-260), all incorporated by reference herein.

For administering to humans, the invention may employ pharmaceuticalcompositions comprising the recombinant adenovirus and apharmaceutically acceptable carrier or excipient. In the presentcontext, the term “pharmaceutically acceptable” means that the carrieror excipient, at the dosages and concentrations employed, will not causeany unwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The purified recombinant adenovirus preferably isformulated and administered as a sterile solution although it is alsopossible to utilize lyophilized preparations. Sterile solutions areprepared by sterile filtration or by other methods known per se in theart. The solutions are then lyophilized or filled into pharmaceuticaldosage containers. The pH of the solution generally is in the range ofpH 3.0 to 9.5, e.g pH 5.0 to 7.5. The recombinant adenovector typicallyis in a solution having a suitable pharmaceutically acceptable buffer.The solution may further also contain a salt. Optionally stabilizingagent may be present, such as albumin. In certain embodiments, detergentis added. In certain embodiments, the recombinant adenovirus may beformulated into an injectable preparation. These formulations containeffective amounts of adenovirus, are either sterile liquid solutions,liquid suspensions or lyophilized versions and optionally containstabilizers or excipients. An adenovirus vaccine can also be aerosolizedfor intranasal administration (see e.g. WO 2009/117134).

For instance, adenovirus may be stored in the buffer that is also usedfor the Adenovirus World Standard (Hoganson et al, Development of astable adenoviral vector formulation, Bioprocessing March 2002, p.43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another usefulformulation buffer suitable for administration to humans is 20 mM Tris,2 mM MgCl₂, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v.Obviously, many other buffers can be used, and several examples ofsuitable formulations for the storage and for pharmaceuticaladministration of purified (adeno)virus preparations can for instance befound in European patent no. 0853660, U.S. Pat. No. 6,225,289 and ininternational patent applications WO 99/41416, WO 99/12568, WO 00/29024,WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

In certain embodiments of the invention, use is made of an adenovirusformulation as described in WO2015/040002. Thus, in a preferredembodiment, the composition comprising the adenovirus vector comprisinga nucleic acid encoding the RSV F protein of SEQ ID NO: 1 comprises inaddition to the recombinant adenovirus; a citrate buffer, wherein thecitrate concentration is ranging between about 5 mM and 30 mM;hydroxypropyl-beta-cyclodextrin (HBCD), wherein the concentration ofHBCD is ranging between about 1% (w/w) and 10% (w/w); a salt, e.g.sodium chloride in a concentration between about 20 mM and about 200 mM;and non-ionic detergent, e.g. Polysorbate-80 in a concentration rangingfrom about 0.005% (w/w) to about 0.5% (w/w); wherein said formulationhas a pH ranging between 5.5 and 6.5.

In certain embodiments, the compositions have a pH ranging between about5.7 and 6.3, and comprise citrate at a concentration ranging betweenabout 5 and 30 mM; HBCD at a concentration ranging between 1% (w/w) and10% (w/w); NaCl at a concentration ranging between 20 mM and 200 mM;Polysorbate-80 at a concentration ranging between about 0.01% (w/w) and0.05% (w/w).

In certain embodiments, the compositions comprise citrate at aconcentration of about 15 mM; HBCD at a concentration of about 5% (w/w);NaCl at a concentration of about 75 mM, and Polysorbate-80 at aconcentration of about 0.03% (w/w).

In certain embodiments, the compositions further comprise ethanol,wherein the ethanol concentration is ranging between about 0.1% (w/w) to1% (w/w).

In a preferred embodiment, the compositions comprise citrate at aconcentration of about 15 mM; HBCD at a concentration of about 5% (w/w);NaCl at a concentration of about 75 mM, Polysorbate-80 at aconcentration of about 0.03% (w/w) and ethanol at a concentration ofabout 0.4% (w/w). The invention is further illustrated in the following,non-limiting, examples.

EXAMPLES Example 1 Stabilizing the RSV F Protein in its Pre FusionConformation

Plasmids encoding basic RSV F sequences were synthesized and the aminoacid substitutions were introduced in the protein by site-directedmutagenesis. The protein variants were transiently expressed in HEK293cells. The relative protein expression on the cell surface was assessedby Flow Cytometry. The stability of the F proteins in pre-fusionconformation was evaluated in a heat-stability assay.

The protein sequence used for RSV A2 F protein variants was retrievedfrom the GenBank, accession number ACO83301.1. The amino acidsubstitutions were introduced in the sequence by site-directedmutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Agilenttechnologies). The mutagenesis primers were designed using on-line toolPrimerX. HEK293T cells (CRL-11268) were purchased from American TissueCulture Collection and cultured under standard cell culture conditions(37° C., 10% CO2).

Fully human IgG1 anti-RSV F protein antibodies CR9501 and CR9503 wereconstructed by cloning the heavy (VH) and light (VL) chain variableregions into a single IgG1 expression vector. PER.C6® cells (Crucell)were transfected with the IgG1 expression constructs and the expressedantibodies were purified from culture supernatants using POROSMabcapture A chromatography (Applied Biosystems) and then bufferexchanged to 50 mM NaAc, 50 mM NaCl, pH 5.5. Antibody concentration wasmeasured by optical absorption at 280 nm. Antibody quality was alsoconfirmed by size-exclusion chromatography (SEC), SDS-PAGE andisoelectric focusing. The antibody CR9501 comprises VH and VL regions of58C5 (as described in WO2011/020079) which binds specifically to RSV Fprotein in its pre-fusion conformation and not to the post-fusionconformation. CR9503 comprises VH and VL regions of motavizumab, whichrecognizes both the pre-fusion and post-fusion conformation of RSV F.

Protein Expression and Temperature Treatment:

The plasmids were transiently transfected into adherent HEK293T cellsusing 293fectine (Cat# 12347-019) transfection reagents (LifeTechnologies) according to suppliers recommendations. 48 hours posttransfection the cells were harvested by detaching with EDTA-containingFACS buffer (no trypsin, see next section) and cell suspension washeat-treated for 10 minutes either in a water bath or in PCR block forthe temperature stability experiments. After the heat-treatment, thecells were prepared for the Flow Cytometry analysis.

For analysis of adeno expressed F proteins, A549 cells were infectedwith Ad26 virus at a MOI of 10 000 or 5000 and Ad35 viruses at a MOI of5000, 2500 or 1.250. After 48 h, the cells were detached and heattreated for 15 minutes at 37° C., 50° C. and 56° C. Upon heat treatmentcells were stained using CR9501-Alexa647 or CR9503-Alexa647 andPropidium Iodide (PI). After staining, the cells were fixed and analyzedusing the BD FACS CANTO II cell analyzer.

Flow Cytometry Analysis:

For each staining, the following controls were included: 1) negativecontrol sample. i.e. cells that were not subjected to any treatment andnot stained with any antibody labeled with a fluorophore; 2) positivecontrol samples, i.e. cells that are stained with only one fluorophore(one of each that are used for the experiment).

The cells were resuspended in the Flow Cytometry (FC Buffer, 5 mM EDTA,1% FBS in PBS) and distributed in volumes of 50 μl of the cellsuspension per well in a 96-well plate with a lid (U- or V-bottomplates). Two-step or one-step protocols were used for staining.

In case of the two-step protocol 50 μl of the first Abs (or buffer fornegative controls) was added to the wells and incubated at RT for 30min.Biotinylated CR9501and CR9503 were used at 2 μg/ml (final concentrationin a well 1 μg/ml). After incubation, the cells were washed 2 times withthe FC buffer. Afterwards 50 μl of Streptavidin-APC (Molecular Probescat#SA1005, 0.1 mg/ml is used at 1:100) or buffer for negative controlswas added to the wells and incubated at RT for 30min. The cells werewashed again 2 times with the FC buffer. After the last wash, the cellswere resuspended in 100 μl of FC buffer +/− live-dead stain (PI fromInvitrogen, cat#P1304MP, used at 2 μg/ml) and incubated at RT for 15minutes. The cells were centrifuged at 200 g (1000 rpm) for 5 min., thebuffer with PI was removed and the cells were resuspended in 150 μl ofthe FC buffer.

In case of a one-step protocol, CR9501 and CR9503 antibodies werelabeled with fluorescent probe Alexa647 (Molecular Probes, cat#A-20186)according to manufacturer's instructions. Cells were stained accordingto the protocol above excluding the Streptavidin-APC step.

From the live cell population, the percentage of cells positive forCR9501/CR9503 antibody binding was determined. The cells positive forCR9503 binding express RSV F protein on their surface. The cellspositive for CR9501 binding express pre-fusion RSV F on their surface.

The intensity of the antibody staining (Median fluorescenceintensity-MFI) is proportional to the amount of F protein on the cellsurface. MFI was calculated from the live cell population expressing Fprotein.

Results: Surface Cell Expression of the Full Length F Protein Variants:

A subset of mutations that was previously identified to increaseexpression or stability of the RSV F protein ectodomain in pre-fusionconformation was introduced in the wild type full length RSV A2 Fsequence (accession number Genbank ACO83301). The mutations wereintroduced alone or in multiple combinations, and the effect on proteinexpression and stability was assessed.

The expression level of the protein was measured as mean fluorescenceintensity (MFI) by Flow Cytometry after staining with the CR9503antibody that is recognizing both pre-fusion and post-fusion F protein.The combination of the two amino acid substitutions that were previouslydescribed for stabilization of the soluble RSV pre-F protein (i.e. N67Iand S215P) also increased the expression level of the full length RSV Fprotein by 2.3-fold, relative to wilde type full length RSV F (FIG. 2).

A prominent increase in expression was observed for variants with 3amino acid substitutions combined. Interestingly, combination of morethan three mutations in one variant did not further increase proteinexpression. This may be due to limited capacity of the cellular membraneto accommodate multiple copies of F protein.

The amount of the pre-fusion F on the surface of the cell was assessedby staining with pre-fusion specific antibody CR9501 (FIG. 3).Transfection of the cells with all F variants resulted in a more or lesssimilar amount of pre-fusion F protein on the cell surface. Presence ofthe transmembrane domain stabilizes the full length protein to certainextent and therefore differences in the pre-fusion stability are not asapparent under ambient conditions between the full length F proteins.Therefore the heat-stability assay was developed to better discriminatestability of full length variants, as decribed below.

The A2 strain that was used as a parental sequence for the previouslydescribed F protein variants (WO2014/174018 and WO2014/202570) is a cellline adapted laboratory strain which has accumulated two unique and raremutations (i.e. of Lysine 66 and Isoleucine 76). In the presentinvention, these two residues were mutated to match the natural clinicalisolates (K66E, I76V). The K66E and I76V mutations were included inselected protein designs. In comparison to variants with Lys66 andIle76, variants with glutamate at 66 (K66E) have a tendency to expressslightly higher. Addition of valine at residue 76 (a double substitutionof K66E and I76V) does not influence expression level when compared tovariants with K66E substitution alone (FIG. 4).

Stability of the Full Length F Protein Variants on the Cell Surface:

In ambient conditions on a short time scale, no significant differencein stability of pre-fusion conformation was observed between full lengthF variants with the different combinations of stabilizing mutations. Anelevated temperature is known to serve as an efficient in vitro triggerfor refolding of RSV F protein from pre-fusion to post-fusionconformation. Therefore, a heat-shock assay was established and used toassess stability of the membrane-bound full length proteins. Shortly,the HEK293T cells were transfected with the F protein constructs andused for the assay 48 hours after transfection. The cells were detachedfrom cell culture dishes and the cell suspension was heat-treated atincreasing temperatures for 10 minutes. After the heat-treatment, cellswere stained with the anti-RSV F antibodies and analyzed by FlowCytometry. The Flow Cytometry data was analyzed in two different ways.The percentage of the cells, positive for staining with the anti-Fantibodies was analyzed, and also mean fluorescence intensity (MFI) ofthe positive cells was calculated (FIG. 5).

Both staining with CR9501 (antibody recognizing only pre-fusion Fprotein) and CR9503 (antibody recognizing both pre- and post-fusion Fprotein) were used in the Flow Cytometry assays. CR9503 antibody servedas a positive control. In case when F protein loses pre-fusionconformation but still is on the surface of the cell, the protein isstill detected with the CR9503 antibody. Loss of staining with bothantibodies indicates that protein is not available on the cell surfacefor antibody binding, e.g. due to aggregation.

Full length proteins with three of more amino acid substitutions weretested in the assay and compared to the wild type RSV F. The expressionof these variants was the highest and therefore these variants werepreferred candidates. All of the proteins contained the N67I and S215Psubstitutions, and one or two extra stabilizing mutations were added.

The unmodified wild type protein had a rather stable staining withCR9503 antibody. The MFI of the CR9503 staining was elevated at highertemperatures however the spread of values was also very high. Thisindicated that no protein aggregation occurred after the heat-shock.Half of the pre-fusion conformation was lost after incubation of cellsat approximately 55° C., after incubation of at 60° C. all pre-fusionconformation was lost as was demonstrated by decreased CR9501 binding tothe wild type F samples after heat-shock at increasing temperatures.

All tested pre-fusion F protein variants were more stable than the wildtype RSV F with majority of the CR9501 staining retaining also aftertreatment at higher temperatures (FIGS. 5 and 6). Proteins with K498Ramino acid substitution were less stable than the others. Addition ofthe K66E mutation further stabilized the proteins as also variants withK498R amino acid substitution became as stable as others and no loss ofthe pre-fusion conformation was observed at 60° C. Only selectedcombinations of the stabilizing mutations were tested with K66E and I76Vcombined. All four tested proteins were stable when percentage ofpositive cells was analyzed, however when MFI was analyzed variant withK498R showed clear decrease in CR9501 binding after treatment with 60°C., indicating that this variant is less stable when evaluated in thetemperature stress assay.

In conclusion, a combination of three stabilizing mutations (includingN67I and

S215P) was considered sufficient for high expression level andstability. The D486N mutations was selected as a third stabilizingmutation because of its position in the protein structure. K66E and I76Vwere included in the as they did not have negative effect on the proteinexpression and stability but made the sequence closer to naturallyoccurring ones.

The pre-fusion RSV F protein with the mutations K66E, N67I, I76V, S215Pand D486N (F2.2) (SEQ ID NO: 1) was selected for the construction ofadenoviral vectors. This protein was shown to be stable in thepre-fusion conformation in the temperature stability assay up to 60° C.,and could be expressed in high levels.

Example 2 Preparation of Adenoviral Vectors

Cloning RSV F Gene into E1 Region of Ad35 and Ad26:

The nucleic acid sequences, coding for the pre-fusion F proteins of theinvention were gene optimized for human expression and synthesized, byGeneart. A

Kozak sequence (5′ GCCACC 3′) was included directly in front of the ATGstart codon, and two stop codons (5′ TGA TAA 3′) were added at the endof the RSV.pre-F coding sequence. The RSV.pre-F genes were inserted inthe pAdApt35BSU plasmid and in the pAdApt26 plasmid via HindIII and XbaIsites.

Cell Culture:

PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9: 1909-1917) weremaintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetalbovine serum (FBS), supplemented with 10 mM MgCl₂.

Adenovirus Generation, Infections and Passaging:

All adenoviruses were generated in PER.C6® cells by single homologousrecombination and produced as previously described (for rAd35: Havengaet al., 2006, J. Gen. Virol. 87: 2135-2143; for rAd26: Abbink et al.,2007, J. Virol. 81: 4654-4663). Briefly, PER.C6 cells were transfectedwith Ad vector plasmids, using Lipofectamine according to theinstructions provided by the manufacturer (Life Technologies). Forrescue of e.g. Ad35 vectors carrying the RSV.pre-F transgenes expressioncassette, the pAdApt35BSU.RSV.pre-F plasmid andpWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, whereas for Ad26 vectorscarrying the RSV.pre-F transgene expression cassette, thepAdApt26.RSV.pre-F plasmid and pWE.Ad26.dE3.5orf6.cosmid were used.Cells were harvested one day after full CPE, freeze-thawed, centrifugedfor 5 min at 3,000 rpm, and stored at −20° C. Next the viruses wereplaque purified and amplified in PER.C6 cultured on a single well of amultiwell 24 tissue culture plate. Further amplification was carried outin PER.C6 cultured using a T25 tissue culture flask and a T175 tissueculture flask. Of the T175 crude lysate, 3 to 5 ml was used to inoculate20×T175 triple-layer tissue culture flasks containing 70% confluentlayers of PER.C6 cells. The virus was purified using a two-step CsClpurification method. Finally, the virus was stored in aliquots at −85°C.

Example 3 Induction of Immunity Against RSV F Using RecombinantAdenovirus Serotypes 26 and 35 Expressing Pre-Fusion RSV F in vivo.

The immunogenicity of Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 wasevaluated in mice, comparing cellular and humoral immune responses toresponses induced by identical doses of Ad26.RSV.FA2 (i.e. expressingthe wild type RSV F protein). Balb/c mice (n=4 per group) were immunizedwith the indicated dose of 10⁸ to 10¹⁰ viral particles (vp) Ad26.RSV.FA2or Ad26.RSV.preF2.1 or Ad26.RSV.preF2.2, or with formulation buffer. At8 weeks after prime, the number of RSV F A2 specific IFNγ spot formingunits (SFU) per 10⁶ splenocytes was determined using ELISpot. It wasshown that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced increasedhumoral immune responses in mice when compared to Ad26.RSV.FA2, withbroad neutralizing capacity and maintained cellular responses. A singleintramuscular immunization with Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2elicited a cellular response (FIG. 7) which was characterized byinduction of CD8+ T cells positive for IFNγ, IL2 and/or TNFα (data notshown).

The quantity and quality of the cellular responses were comparablebetween Ad26.RSV.preF2.1, Ad26.RSV.preF.2.2 and Ad26.RSV.FA2. Incontrast, Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced significantlyhigher RSV neutralizing antibody titers than Ad26.RSV.FA2. Closeranalysis of the antibody responses demonstrated that Ad26.RSV.preF2.1and Ad26.RSV.preF.2.2 induced higher levels of antibodies againstpre-fusion F, while post-fusion F titers remained comparable toAd26.RSV.FA2, resulting in significantly increased preF/postF antibodyratios. In addition, the IgG2a/IgG1 ratio of the antibody responseremained unaltered, demonstrating a similar Th1 skewing of the humoralresponse as previously demonstrated for Ad26.RSV.FA2 (FIG. 8).

For Ad26.RSV.preF2.2 it was furthermore demonstrated that the antibodieselicited were capable of neutralizing various RSV A and B strains,laboratory strains as well as clinical isolates, similar as observed forAd26.RSV.FA2 (FIG. 9).

Subsequently, the efficacy and immunogenicity of Ad26.RSV.preF2.2 andAd35.RSV.preF2.2 vector constructs was evaluated in the cotton ratmodel. These animals are permissive to replication of human RSV, withpeak RSV titers in the lungs at days 4 and 5. Control groups in theexperiments included groups intranasally infected with a low dose RSVA2, thereby mimicking natural exposure, as well as groups immunized withFI-RSV, using the original lot 100 that induced enhanced respiratorydisease (ERD) in clinical studies in the dilution that was shown toinduce ERD in cotton rats.

Single intramuscular immunization of animals with Ad26.RSV.preF2.2 indoses ranging from 10⁵ to 10⁸ vp/animal, or Ad35.RSV.preF2.2 in dosesranging from 10⁶ to 10⁹ vp/animal resulted in complete protection of thelungs from infection with the vaccine homologous RSV A2 strain, exceptfor 3 animals immunized with 10⁵ vp Ad26.RSV.preF2.2 (FIG. 10A and 10B).Dose dependent protection of RSV replication in the nose was observedfor both vectors. This ranged from full protection at 10⁸ vp/animal, topartial protection at 10⁵ vp for Ad26.RSV.preF2.2, whereas forAd35.RSV.preF2.2, noses of animals immunized with 10⁹ vp were fullyprotected from RSV A2, and 10⁶ vp resulted in partial protection (FIG.10C and 10D) Noses of animals immunized with Ad26.RSV.preF2.2 andAd35.RSV.preF2.2 were better protected from RSV A2 infection than whenimmunized with their respective wild type F counterparts Ad26.RSV.FA2and Ad35.RSV.FA2, when analyzed across dose (p=0.0003, and p=0,0001).Protection from RSV infection was accompanied by dose-dependentinduction of virus neutralization titers against RSV A Long, alreadyelicited by the lowest doses of Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2applied (FIG. 10E and 10F). Across dose statistical comparisons of VNA ALong titers revealed that Ad26.RSV.preF2.2 is more immunogenic thanAd26.RSV.FA2 (p=0.0414), whereas elicitation of VNA titers was notsignificantly different between Ad35.RSV.preF2.2 and Ad35.RSV.FA2.

It was further demonstrated that Ad26.RSV.preF and Ad35.RSV.preF do notinduce histopathological signs of Enhanced Respiratory Disease (ERD)after RSV A2 challenge, at any of the concentrations tested. The cottonrat is the most used and best studied model to monitor ERD. In thisanimal model, vaccination with FI-RSV consistently induces ERD after RSVchallenge, which is visible by histopathological analysis of sections ofthe infected lungs for parameters as alveolitits, consisting primarilyof neutrophil infiltrates, and peribronchiolitis, consisting primarilyof lymphocyte infiltrates. In cotton rats, FI-RSV-induced scores forthese parameters can be observed from day 1 after RSV infection, andpeak at 4 to 5 days after RSV challenge.

ERD was analyzed 5 days after challenge with RSV A2 by scoring 4parameters of pulmonary inflammatory changes (peribronchiolitis,perivasculitis, interstitial pneumonia, alveolitis). Immunization withFI-RSV resulted in enhanced scores for most histopathological markers,which was especially apparent for alveolitis (FIG. 11), the marker thatwas previously shown to be the most discriminating marker for ERD. Noincreases in alveolitis or any other ERD histopathological marker wasobserved in animals immunized by either Ad26.RSV.preF2.2 orAd35.RSV.preF2.2 in a prime-only regimen after RSV challenge, even atlow vaccine doses that may induce low affinity and/or low levels ofantibodies (FIG. 11). This is confirming our previous results withAd26.RSV.FA2 and Ad35.RSV.FA2 vectors.

According to the invention, it has thus been shown that Ad26.RSV.preF2.2and Ad35.RSV.preF2.2 are potent adenoviral vectors expressing RSV F A2which is stabilized in the pre-fusion conformation. These vectors inducestrong humoral and cellular immune responses. The immune responseelicited is protective against RSV A2 challenge and provides a widerange of virus neutralization in vitro against clinical and laboratoryisolates of RSV. No ERD induction was observed in cotton rats after RSVexposure of vaccinated animals and therefore confirms the data generatedwith Ad26 and Ad35 encoding for the wild type RSV F A2 antigen. Neithermice nor cotton rats showed overt signs of reactogenicity afterinjection of either Ad26.RSV.preF2.2 or Ad35 .RSV.preF2.2.

Example 4 Study of RSV Vaccine Recombinant Adenovirus Serotypes 26Expressing Pre Fusion RSV F in Humans

A Randomized, Double-blind, First-in-Human Phase 1 Study to evaluate thesafety, tolerability and immunogenicity of two Vaccinations ofAd26.RSV.preF, one year apart, in adults aged 60 years and older instable health was performed. This trial has been registered atclinicaltrials.gov identifier NCT02926430.

Objectives:

The primary objective of the study included assessing the safety andtolerability of 2 single doses of either 5×10¹⁰ viral particles [vp] or1×10¹¹ vp of Ad26.RSV.preF, administered intramuscularly to adults above60 years of age. The secondary objective included assessing the humoraland cellular immune responses as measured by virus neutralization assay(VNA), F-protein binding antibodies (pre-F and post-F ELISA) andintracellular cytokine staining (ICS).

Study Design

This is a single-center, randomized, placebo-controlled, double-blind,Phase 1 study to evaluate the safety, tolerability and immunogenicity of2 Ad26.RSV.preF vaccinations, administered 1 year apart, in 72 male andfemale subjects aged 60 years and older in stable health. Subjects wererandomized in parallel in a 1:1:1:1:2 ratio to 1 of 5 groups to receive2 single intramuscular (IM) injections of study vaccine 12 monthsapart(Table 1).

Safety Results:

An interim analysis at 28 days after first vaccination was performed.Subjects that received 5×10¹⁰ vp Ad26.RSV.preF on Day 1, were pooledtogether (Group 1 and 2), the same for subjects that received 1×10¹¹ vpAd26.RSV.preF (Group 3 and 4). The primary interim analysis post firstdose of all 72 subjects demonstrates that this vaccine is well toleratedat both dose levels. The median duration of solicited adverse events(AEs) typically ranged from 1 to 3.5 days. There was no serious AErelated (SAE) to the vaccine, and no AEs led to withdrawal from thestudy. There was no apparent difference in reactogenicity between thetwo vaccine doses used. The interim data did not reveal an effect ofdose on the safety.

In conclusion, overall solicited and unsolicited AEs reportedpost-vaccination were mild in the majority of subjects, transient innature, and resolved without sequelae. Based on the above, it wasconcluded that both doses of Ad26.RSV.preF were safe and well-toleratedby participants of both dosage groups.

Immunogenicity Assays Pre-F ELISA

Total IgG levels against the pre-F stabilized conformation (based on RSVA2 F, Genbank ACO83301.1) were evaluated. The pre-F protein wasbiotinylated and sequentially captured by streptavidin coated plates on96-well microtiter plates. Serially diluted test samples were incubatedand pre-F-specific antibodies were detected with anti-human IgGantibodies conjugated with horseradish peroxidase followed by achemiluminescent reaction. The IC₅₀ was reported as the binding titer.

Post-F ELISA

Total IgG levels against the post-F protein (RSV A2 F, GenbankACO83301.1) were evaluated. The post-F protein was coated on 96-wellmicrotiter plates. Serially diluted test samples were incubated andpost-F-specific antibodies were detected with anti-human IgG antibodiesconjugated with horseradish peroxidase followed by a chemiluminescentreaction. The IC₅₀ was reported as the binding titer.

RSV Neutralization Assay A2 Strain (A2-FFL) or B Strain(A2-BAGdup-036634.1F-FFL)

The functionality of the vaccine-induced antibody responses wasinvestigated by the determination of virus neutralizing antibodies (VNA)in a virus neutralization assay using A549 cells and RSV A2 virus whichexpresses luciferase (Luc) (RSV A2 FFL or RSV B FFL). Neutralizingantibodies were measured in A549 cells as a function of reduction infirefly Luc reporter gene expression after a single round of infectionwith RSV A2-FFL (or B Gdup-FFL). A fixed amount of RSV A2 FFL (or BGdup-FFL) was mixed with a serially diluted clinical serum sample. After1 hour incubation, A549 cells were added to the mixture. RSV A2-FFL (orB Gdup FFL) infection or inhibition was measured at 20-21 hours by theLuc reporter gene expression system. IC50 was reported as theneutralization titer.

ELISpot

Frozen PBMCs were analyzed by IFNγ ELISpot. PBMCs were stimulated withpeptide pools matched to the preF protein vaccine insert (SEQ ID NO: 1).The number of SFC/10⁶ stimulated PBMCs, after subtraction of mockstimulated PBMCs was reported.

CD4+ and CD8+ T-cell Responses (ICS)

The induction or boosting of CD4 and CD8 T-cell subsets expressing IFNγ,IL-2 and TNFα was determined by ICS after RSV F-protein peptidestimulation. Total cytokine responses were reported as the percentage ofCD4+ and CD8+ T cells that produce at least IFNγ, IL-2 and/or TNFα. Forthe Th1/Th2 balance, the CD4 cells expressing either IFNγ (Th1) or IL-4(Th2) was reported . The total responding (log₁₀ scale) CD4+ or CD8+ Tcells, expressing IFNγ, IL-2, TNFα as a single cytokine or in anycombination thereof are added and reported

Immunogenicity Results

The humoral immunogenicity analysis included the following primaryimmuno assays (secondary objective): VNA to the RSV A2 strain andbinding ELISA to pre-fusion and post-fusion F protein. In addition, aVNA to an RSV B strain was performed. Cellular immunogenicity analysisincluded IFNγ ELISPOT assay. The results are shown in Tables 2-7.

As expected, RSV F specific humoral and cellular responses were detectedin all subjects at baseline (day 1), reflecting a history of naturalexposure to RSV throughout life (Tables 2-5).

Table 2 includes the results of VNA responses against RSV A2 strain,presented in descriptive statistics of the actual values and foldincrease from baseline with corresponding 95% Confidence interval (CI)The geometric mean titer (GMT) fold rise for RSV A2 neutralizingantibodies after 1 dose of Ad26.RSV.preF at 5×10¹⁰ or 1×10¹¹ vp was 2.5and 3.2, respectively.

The results of VNA responses against RSV B strain are shown in Table 3,which also includes descriptive statistics of the actual values and foldincrease from baseline with corresponding 95% CI.

The GMT fold rise for RSV B neutralizing antibodies after 1 dose ofAd26.RSV.preF at 5×10¹⁰ or 1×10¹¹ vp was 3.1 and 3.4, respectively.Thus, both RSV A2 and RSV B neutralizing antibodies increased aftervaccination at day 28 post vaccination. Ad26.RSV preF at a dose of1×10¹¹ vp resulted in higher neutralizing antibody levels compared tothe dose of 56×10¹⁰.

Pre-F binding ELISA assessed the binding of antibodies to the pre-fusionconformation of the RSV F protein, while post-F binding ELISA assessedthe binding of antibodies to the post-fusion conformation of the RSV Fprotein. Descriptive statistics of the actual values and fold increasefrom baseline with corresponding 95% CI of the results are presented inTable 2 for both pre-F protein binding antibody responses (ELISA) andpost-F protein binding antibody responses (ELISA). Total preF IgGbinding antibody showed GMT fold rises of 2.2 (5×10¹⁰ vp) and 2.9(1×10¹¹ vp), while total postF IgG binding antibody showed GMT foldincreases of 1.9 (5×10¹⁰ vp) or 2.1 (1×10¹¹ vp) fold. Thus, both thelevel of binding antibodies to F protein in the pre-fusion conformation(preF antibodies) and of post F antibodies increased after one vaccinedose. The results of the preF binding ELISA followed the same pattern asthe VNA assay. The highest levels of preF binding antibodies wasobserved in the group receiving Ad26.RSV.preF at a dose of 1×10¹¹ vp. Nodose response was observed on the induction of postF antibodies using5×10¹⁰ vp or 1×10¹¹ vp Ad26.RSV.preF dose.

T-cell responses induced by Ad26.RSV.preF are measured by IFNγELISpot,in which PBMC are restimulated with the preF protein peptides.Descriptive statistics of the actual values of RSV-F specific T cellresponse (IFNγ) before and after vaccination are presented in Error!Reference source not found. Median cellular responses in these elderlysubjects as measured by IFNγ ELISPOT increased from 103 and 95 atbaseline to 325 and 305 SFU/10⁶ PBMC (5×10¹⁰ and 1×10¹¹ vp,respectively). The total responding CD4+ T cells, expressing IFNγ, IL-2,TNFα as a single cytokine or in any combination thereof are added andpresented in Table 5. An increase in the CD4+ T-cell response wasobserved at 28 days after vaccination with A26.RSV.preF: the mediantotal cytokine response was 0.03% and 0.03% at day 1 compared to 0.13%and 0.12% at day 28 post immunization (5×10¹⁰ and 1×10¹¹ vp,respectively). The total responding CD8+ T cells, expressing-* IFNγ,IL-2, TNFα as a single cytokine or in any combination thereof are alsopresented in Table 5 , the median total cytokine response was 0.07% and0.04% at day 1 compared to 0.15% and 0.06% at day 28 post immunization(5×10¹⁰ and 1 ×10¹¹ vp, respectively).

As shown in Table 6, administration of Ad26.preF changed the ratiobetween antibodies binding to pre-fusion F (preF antibodies) andantibodies binding to post-fusion F (postF antibodies). A geometric meanincrease in preF/postF ratio in the Ad26.preF vaccinated subjects wasseen from 1.5 and 1.2 at baseline to 1.7 to 1.8 (5×10¹⁰ and 1×10¹¹ vp,respectively).The administration of Ad26.RSV.preF thus clearly favoureda higher rise of preF binding antibodies.

The geometric mean preF/VNA ratio in the Ad26.preF vaccinated subjectswas similar for both groups (5×10¹⁰ and 1×10¹¹ vp): 0.8 at baseline and0.7 at 28 days after vaccination (Table 7), while geometric meanpostF/VNA ratio in the Ad26.preF vaccinated subjects was 0.5 and 0.6 atbaseline and 0.4 and 0.4 (5×10¹⁰ and 1×10¹¹ vp, respectively) at day 29(Table 8).

In previously conducted clinical trials VAC18192RSV1001(ClinicalTrials.gov Identifier: NCT02440035) and VAC18192RSV1003(ClinicalTrials.gov Identifier: NCT02561871) with the prototype Ad26.RSVFA2 RSV expressing the wild type RSV F from RSV A2, healthy subjects of18-50 years of age received Ad26.RSV.FA2 at 5×10¹ vp dose. Both humoraland cellular immune responses were increased 28 days after receivingAd26.RSV.FA2. The geometric mean fold rise for RSV neutralizingantibodies was 2.0 and 1.9 for RSV A2 and RSV B respectively. The meanfold rise of total postF IgG binding antibody showed geometric meanincreases of 3.0 fold, while the geometric mean fold rise of preF IgGbinding showed a 2.4 fold rise. Median cellular responses in thesesubjects as measured by IFNγ ELISPOT increased from 76 to 290 SFU/10⁶PBMC (data not shown). In these clinical trials with Ad26.RSV.FA2 anincrease in preF/postF ratio was not observed. More strikingly, thegeometric mean preF/post F ratio was 1.4 at baseline and decreased to1.1 at 28 days after vaccination. The geometric mean preF/VNA ratio was0.6 at baseline and 0.7 at 28 days after vaccination; while thegeometric mean postF/VNA ratio was 0.4 at baseline and increasedslightly to 0.6 at 28 days after vaccination (Tables 10-12).

In conclusion, according to the present invention it was shown that bothhumoral and cellular immune responses were strongly increased 28 daysafter the first vaccine dose for both dose levels of Ad26.RSV.preF (i.e.recombinant adenovirus encoding the RSV F protein in the pre-fusionconformation, in particular the RSV F protein of SEQ ID NO: 1). Thehighest humoral responses were observed with 1×10¹¹ vp dose, which wasstill safe. The boost in humoral immune response was preferably directedagainst the pre-F epitopes, as illustrated by a shift in the preF/postFbinding antibody ratio from baseline. One dose of Ad26.RSV.preF was safeand well tolerated in this phase 1 study and boosted favourable humoraland cellular responses in RSV experienced older adults, even at a higherdose of 1×10¹¹ vp of adenovirus.

TABLE 1 Study design Group N Day 1 Day 365^(a) 1 12 Ad26.RSV.preF (5 ×10¹⁰ vp) Ad26.RSV.preF (5 × 10¹⁰ vp) 2 12 Ad26.RSV.preF (5 × 10¹⁰ vp)Placebo 3 12 Ad26.RSV.preF (1 × 10¹¹ vp) Ad26.RSV.preF (1 × 10¹¹ vp) 412 Ad26.RSV.preF (1 × 10¹¹ vp) Placebo 5 24 Placebo Placebo ^(a)Foroperational reasons, the second vaccination may occur between 12-14months after the first vaccination

TABLE 2 Descriptive Statistics of the Humoral immunogenicity Assays;Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp) (1 × 10¹¹vp) Placebo Humoral: ELISA preF Baseline 24 24 23 DAY 1 N 24 24 23Geometric mean (95% CI) 334 (255; 438) 407 (293; 566) 269 (212; 341)Post-dose 1 23 24 23 DAY 29 N 23 24 23 Geometric mean (95% CI) 709 (519;967) 1193 (897; 1588) 255 (203; 321) Geometric mean increase (95% CI)2.2 (1.7; 2.8) 2.9 (2.2; 3.9) 0.9 (0.9; 1.0) Humoral: ELISA postFBaseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95% CI) 219 (172;279) 326 (228; 466) 174 (132; 231) Post-dose 1 23 24 23 DAY 29 N 23 2423 Geometric mean (95% CI) 407 (305; 543) 680 (509; 908) 170 (129; 224)Geometric mean increase (95% CI) 1.9 (1.5; 2.3) 2.1 (1.8; 2.4) 1.0 (0.9;1.0) Humoral: RSV A2 strain Baseline 24 24 23 DAY 1 N 24 24 23 Geometricmean (95% CI) 432 (328; 569) 512 (345; 759) 402 (280; 578) Post-dose 123 24 23 DAY 29 N 23 24 23 Geometric mean (95% CI) 1031 (839; 1267) 1617(1126; 2323) 431 (299; 622) Geometric mean increase (95% CI) 2.5 (1.9;3.2) 3.2 (2.3; 4.2) 1.1 (1.0; 1.1) N: number of subjects with data

TABLE 3 Titers of Neutralizing Antibodies to RSV B strain: DescriptiveStatistics of the Actual Values and Fold Increase from Baseline; FullAnalysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp) (1 × 10¹¹ vp)Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95% CI) 1787(1252; 2551) 2710 (1678; 4376) 1632 (1065; 2502) Post-dose 1 23 24 23DAY 29 N 23 24 23 Geometric mean (95% CI) 5383 (3770; 7686) 9302 (6273;13795) 1556 (1073; 2257) Geometric mean increase (95% CI) 3.1 (2.1; 4.7)3.4 (2.3; 5) 1 (0.8; 1.1) N: number of subjects with data

TABLE 4 ELISpot: Descriptive Statistics of the Actual Values; FullAnalysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp) (1 × 10¹¹ vp)Placebo Baseline  23  21  21 DAY 1 N  23  21  21 Median (Q1; Q3) 103(42; 175)  95 (37; 245) 114 (42; 169) Post-dose 1  23  21  22 DAY 29 N 23  21  22 Median (Q1; Q3) 325 (249; 478) 305 (172; 492)  94 (61;2 23)N: number of subjects with data

TABLE 5 Total Cytokine Response (ICS): Descriptive Statistics of theActual Values; Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰vp) (1 × 10¹¹ vp) Placebo CD4 Baseline 20 20 22 DAY 1 N 20 20 22 Median0.03 (<0.02; 0.07) 0.03 (<0.02; 0.10) 0.02 (<0.02; 0.06) (Q1; Q3)Post-dose 1 19 19 20 DAY 29 N 19 19 20 Median 0.13 (0.08; 0.16) 0.12(0.05; 0.18) <0.02 (<0.02; 0.04) (Q1; Q3) CD8 Baseline 20 20 22 DAY 1 N20 20 22 Median 0.07 (0.02; 0.20) 0.04 (<0.02; 0.12) 0.09 (<0.02; 0.31)(Q1; Q3) Post-dose 1 19 19 20 DAY 29 N 19 19 20 Median 0.15 (0.08; 0.29)0.06 (0.03; 0.20) 0.10 (0.03; 0.66) (Q1; Q3) N: number of subjects withdata

TABLE 6 Ratio Antibody Response RSV Pre F-protein versus Post F-proteinover Time; Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp)(1 × 10¹¹ vp) Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean1.5 (1.3; 1.8) 1.2 (1; 1.6) 1.5 (1.3; 1.8) (95% CI) Post-dose 1 23 24 23DAY 29 N 23 24 23 Geometric mean 1.7 (1.5; 2.1) 1.8 (1.4; 2.3) 1.5 (1.3;1.8) (95% CI) N: number of subjects with data

TABLE 7 Ratio Antibody Response RSV Pre F-protein versus VNA A2 overTime; Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp) (1 ×10¹¹ vp) Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95%CI) 0.8 (0.6; 0.9) 0.8 (0.7; 1.0) 0.7 (0.5; 0.9) Post-dose 1 23 24 23DAY 29 N 23 24 23 Geometric mean (95% CI) 0.7 (0.5; 0.9) 0.7 (0.6; 0.9)0.6 (0.4; 0.8) N: number of subjects with data

TABLE 8 Ratio Antibody Response RSV Post-F-protein versus VNA A2 overTime; Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5 × 10¹⁰ vp) (1 ×10¹¹ vp) Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95%CI) 0.5 (0.4; 0.6) 0.6 (0.5; 0.8) 0.7 (0.5; 0.9) Post-dose 1 23 24 23DAY 29 N 23 24 23 Geometric mean (95% CI) 0.4 (0.3; 0.5) 0.4 (0.3; 0.6)0.6 (0.4; 0.8) N: number of subjects with data

TABLE 9 Descriptive Statistics of all Humoral Assays; ImmunogenicityAnalysis Set (Study VAC18192RSV1001 + VAC18192RSV1003) Ad26.RSV.FA2, (5× 10¹⁰ vp) Humoral: ELISA pre Baseline 35 DAY 1 N 35 Geometric mean (95%CI) 248 (203; 304) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean(95% CI) 596 (493; 720) Geometric mean increase (95% CI) 2.4 (2.0; 2.9)Humoral: ELISA post Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 184(151; 224) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95% CI)556 (445; 695) Geometric mean increase (95% CI) 3.0 (2.5; 3.7) Humoral:RSV A2 strain Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 433 (345;543) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95% CI) 856(675; 1085) Geometric mean increase (95% CI) 2.0 (1.7; 2.4) Humoral: RSVB strain Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 2231 (1851;2689) Post-Ad.RSV.FA2 dose 23 DAY 29 N 23 Geometric mean (95% CI) 3983(3206; 4948) Geometric mean increase (95% CI) 1.9 (1.6; 2.1) N: numberof subjects with data

TABLE 10 Ratio Antibody Response RSV Pre F-protein versus Post F-proteinover Time; Immunogenicity Analysis Set (Study VAC18192RSV1001 +VAC18192RSV1003) Ad26.RSV.FA2, (5 × 10¹⁰ vp) Baseline 35 DAY 1 N 35Geometric mean (95% CI) 1.4 (1.2; 1.6) Post-Ad.RSV.FA2 dose 35 DAY 29 N35 Geometric mean (95% CI) 1.1 (0.9; 1.2) N: number of subjects withdata

TABLE 11 Ratio Antibody Response RSV Pre F-protein versus VNA A2 overTime; Immunogenicity Analysis Set (Study VAC18192RSV1001 +VAC18192RSV1003) Ad26.RSV.FA2, (5 × 10¹⁰ vp) Baseline 35 DAY 1 N 35Geometric mean (95% CI) 0.6 (0.5; 0.7) Post-Ad.RSV.FA2 dose 35 DAY 29 N35 Geometric mean (95% CI) 0.7 (0.6; 0.9) N: number of subjects withdata

TABLE 12 Ratio Antibody Response RSV Post-F-protein versus VNA A2 overTime; Immunogenicity Analysis Set (Study VAC18192RSV1001 +VAC18192RSV1003) Ad26.RSV.FA2, (5 × 10¹⁰ vp) Baseline 35 DAY 1 N 35Geometric mean (95% CI) 0.4 (0.3; 0.5) Post-Ad.RSV.FA2 dose 35 DAY 29 N35 Geometric mean (95% CI) 0.6 (0.5; 0.8) N: number of subjects withdata

Sequence listing Amino acid sequence of the RSV pre-fusion Fprotein (mutations compared to RSV A2 strain are bold and underlined)SEQ ID NO: 1: RSV preF2.2 amino acid sequence:MELLILKANA ITTILTAVTF CFASGQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE LSNIKEI KCN GTDAK V KLIK QELDKYKNAV TELQLLMQST PATNNRARRE LPRFMNYTLNNAKKTNVTLS KKRKRRFLGF LLGVGSAIAS GVAVSKVLHLEGEVNKIKSA LLSTNKAVVS LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSI PNIETV IEFQQKNNRL LEITREFSVN AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQIVRQQSYSIMS IIKEEVLAYV VQLPLYGVID TPCWKLHTSPLCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKVQSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKTDVSSSVITSL GAIVSCYGKT KCTASNKNRG IIKTFSNGCDYVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP LVFPS NEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAVKSTTNIMITTI IIVIIVILLS LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSNSEQ ID NO: 2: codon optimized nucleic acidencoding the RSV F pre-F2.2 pre-fusion proteinATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCTGCAGCGCCGTGAGCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAGGAGATCAAGTGCAACGGCACCGACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACAGAGCCAGAAGAGAGCTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACCAACGTGACCCTGAGCAAGAAGAGAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATCGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGGCGAGGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACAGACTGCTGGAGATCACCAGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAGAAGCTGATGAGCAACAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATCATGAGCATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAGGAGGGCAGCAACATCTGCCTGACCAGAACCGACAGAGGCTGGTACTGCGACAACGCCGGCAGCGTGAGCTTCTTCCCCCAGGCCGAGACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATGACCAGCAAGACCGACGTGAGCAGCAGCGTGATCACCAGCCTGGGCGCCATCGTGAGCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACAGAGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTGAGCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAACGTGAACGCCGTGAAGAGCACCACCAACATCATGATCACCACCATCATCATCGTGATCATCGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGAAGCACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTCAGCAACTGA

1. A method of inducing a safe immune response against respiratorysyncytial virus (RSV) in a human subject in need thereof, comprisingadministering to the subject a composition comprising recombinantadenovirus comprising nucleic acid encoding an RSV Fusion (F) proteincomprising the amino acid sequence of SEQ ID NO: 1, and apharmaceutically acceptable carrier, in a total dose of from about1×10¹⁰ to about 2×10¹¹ viral particles (vp).
 2. The method according toclaim 1, wherein the compositions comprises recombinant adenoviruscomprising nucleic acid encoding an RSV Fusion (F) protein comprisingthe amino acid sequence of SEQ ID NO: 1, and a pharmaceuticallyacceptable carrier, in a total dose of from about 5×10 to about 1×10¹¹viral particles (vp).
 3. The method according to claim 1, wherein theimmune response comprises the induction of antibodies specificallybinding to RSV F protein.
 4. The method according to claim 1, whereinthe immune response comprises the induction of RSV neutralizingantibodies.
 5. The method according to claim 1, wherein the immuneresponse comprises the induction of antibodies specific for the RSV Fprotein in the pre-fusion conformation and antibodies specific for theRSV F protein in the post-fusion conformation, and wherein the geometricmean titer (GMT) increase of antibodies specific for RSV F protein inthe pre-fusion conformation is higher than the geometric mean titer(GMT) increase of antibodies specific for RSV F protein in thepost-fusion conformation, in enzyme linked immunosorbent assays(ELISAs).
 6. The method according to claim 1, wherein the ratio betweenthe geometric mean titer (GMT) increase of post-fusion F specificantibodies as measured in ELISA and the geometric mean titer (GMT)increase of neutralizing antibodies as measured in a VNA assay isreduced after administration of said composition as compared to saidratio before administration of said composition.
 7. The method accordingto claim 1, wherein the immune response further comprises a cellularresponse as indicated by IFNgamma producing T cells as measured in anIFNy ELISPOT in response to stimulation with a pool of peptides coveringthe RSV F protein of SEQ ID NO: 1, and/or by measurement of CD4 and CD8T-cell subsets expressing IFNγ, IL-2 and TNFα by intracellular staining(ICS) after stimulation with a pool of peptides covering the RSV Fprotein of SEQ ID NO:
 1. 8. The method according to claim 1, wherein thesubject is a human of 60 years or older.
 9. The method according toclaim 1, wherein the nucleic acid encoding the RSV F protein comprisesthe nucleic acid sequence of SEQ ID NO:
 2. 10. The method according toclaim 1, wherein the recombinant adenovirus is a human adenovirus. 11.The method according to claim 10, wherein the adenovirus is of serotype26 or 35.